During the current funding period we demonstrated the significant anti-leukemic potential of adoptively transferred adult NK cells by effecting complete remissions in patients with advanced AML. This project will include novel clinical trials designed to improve techniques exploiting NK cell alloreactivity in umbilical cord blood (UCB) transplants as well as in vitro and murine experiments to characterize the mechanisms supporting the expansion and education of NK cells derived from adult blood and from UCB progenitors. In SA1, we will perform a series of clinical trials assessing the efficacy of IL-2 administration to enhance in vivo NK cell expansion and education in an adult NK cell adoptive transfer/T cell depleted UCB transplantation strategy. We have generated in vitro data suggesting an important effect of IL-15 on NK cell expansion and differentiation. Clinical grade IL-15 has recently been made available to us by the NCI, and we will next substitute IL-15 for IL-2 in the first human trial to test the ability of IL-15 to facilitate education of engrafting NK cells after UCB transplant. A third clinical trial will test the hypothesis that adoptive transfer of >10 fold higher doses of ex vivo expanded adult NK cells will further enhance efficacy. In SA2 we will monitor the receptor repertoires, function and education of NK cells expanding in patients exposed to IL-2 or IL-15, after Treg infusion from the UCB transplant trials in Project 1 (Wagner), and in transplant recipients responding to viral stress. We will also use pre-clincial in vitro assays and a murine model in which adult human NK cells and UCB progenitors are transferred into NOD SCID IL2Rgamma-c-null immunodeficient mice to test novel strategies to enhance NK cell function. Specifically, we will test methods to enhance the anti-leukemic function of NK cells with potential for translation into clinical trials. We will test trans-presentation of IL-15 using lL-15Ra-Fc, ex vivo NK cell expansion using IL-15Ra+ artificial K562 cell stimulator cells ([unreadable] Notch ligand or41BB ligand), and techniques to tailor the allo-reactive specificity of ex vivo expanded NK cells by culture with K562 stimulators expressing single Bw4, C1 or C2 KIR ligands. Ultimately, the best strategy from this series of clinical trials designed to optimize the anti-leukemic effect of NK cells will be combined with the clinical methods to enhance immune reconstitution and minimize GVHD developed in Project 1 and in Project 2.